SummaryThe immutans (ira) variegation mutant of Arabidopsis thaliana contains green-and white-eactored leaves due to the action of a nuclear recessive gene. The mutation is somatically unstable, and the degree of sectoring is Influenced by light and temperature. Whereas the cells in the green sectors contain normal chloroplasts, the cells in the white sectors are heteroplasUdic and contain non-pigmented plastids that lack organized lamellar structures, as well as small pigmented plastids and/or rare normal chloroplasts. This indicates that the plastids in im white cells are not affected equally by the nuclear mutation and that the expression of irnmutans is 'plastid autonomous'. In contrast to other variegation mutants with heteroplastidic cells, the defect in im is not maternally Inherited. immutans thus represents a novel type of nuclear gene-induced variegation mutant. It has also been found that the white tissues of immutans accumulate phytoene, a non-colored C4o carotenoid intermediate. This suggests that immutans controls, either directly or indirectly, the activity of phytoene desaturaea (PDS), the enzyme that converts phytoene to zeta-carotene in higher plants. However, im is not the structural gene for PDS. A secondary effect of carotenoid deficiency, both in immutans and in wildtype plants treated with a herbicide that blocks carotenoid synthesis, is an increase in acid ribonuclease activity in white tissue. It is concluded that the novel variegation generated by the immutans mutation should offer great insight into the complex circuitry that regulates nuclear--organelle interactions.

Nuclear gene-induced variegation mutants provide a powerful system to dissect interactions between the genetic systems of the nucleus-cytoplasm, the chloroplast, and the mitochondrion. The immutans (im) variegation mutation of Arabidopsis is nuclear and recessive and results in the production of green- and white-sectored leaves. The green sectors contain cells with normal chloroplasts, whereas the white sectors are heteroplastidic and contain cells with abnormal, pigment-deficient plastids as well as some normal chloroplasts. White sector formation can be promoted by enhanced light intensities, but sectoring becomes irreversible early in leaf development. The white sectors accumulate the carotenoid precursor phytoene. We have positionally cloned IM and found that the gene encodes a 40.5-kD protein with sequence motifs characteristic of alternative oxidase, a mitochondrial protein that functions as a terminal oxidase in the respiratory chains of all plants. However, phylogenetic analyses revealed that the IM protein is only distantly related to these other alternative oxidases, suggesting that IM is a novel member of this protein class. We sequenced three alleles of im, and all are predicted to be null. Our data suggest a model of variegation in which the IM protein functions early in chloroplast biogenesis as a component of a redox chain responsible for phytoene desaturation but that a redundant electron transfer function is capable of compensating for IM activity in some plastids and cells.

Nuclear gene-induced variegation mutants provide a powerful system to dissect interactions between the genetic systems of the nucleus-cytoplasm, the chloroplast, and the mitochondrion. The immutans ( im ) variegation mutation of Arabidopsis is nuclear and recessive and results in the production of green-and white-sectored leaves. The green sectors contain cells with normal chloroplasts, whereas the white sectors are heteroplastidic and contain cells with abnormal, pigment-deficient plastids as well as some normal chloroplasts. White sector formation can be promoted by enhanced light intensities, but sectoring becomes irreversible early in leaf development. The white sectors accumulate the carotenoid precursor phytoene. We have positionally cloned IM and found that the gene encodes a 40.5-kD protein with sequence motifs characteristic of alternative oxidase, a mitochondrial protein that functions as a terminal oxidase in the respiratory chains of all plants. However, phylogenetic analyses revealed that the IM protein is only distantly related to these other alternative oxidases, suggesting that IM is a novel member of this protein class. We sequenced three alleles of im , and all are predicted to be null. Our data suggest a model of variegation in which the IM protein functions early in chloroplast biogenesis as a component of a redox chain responsible for phytoene desaturation but that a redundant electron transfer function is capable of compensating for IM activity in some plastids and cells. INTRODUCTIONVariegation mutants have played a prominent role in the history of genetics (reviewed in Granick, 1955). As a notable example, they were used by E. Bauer in his seminal studies in the early 1900s to describe the phenomenon of non-Mendelian inheritance. The cells in the green sectors of these plants have morphologically normal chloroplasts, whereas cells in the white sectors have abnormal plastids deficient in pigments and organized lamellar structures. One common mechanism of variegation involves the induction of defective mitochondria or chloroplasts in some but not all cells by mutations in nuclear genes (Tilney-Bassett, 1975). Because the products defined by these genes are required for normal chloroplast biogenesis, they provide an excellent starting point to dissect the poorly understood pathways of communication between the nuclear-cytoplasmic, chloroplast, and mitochondrial genetic systems (Taylor, 1989).Several well-known examples of variegated plants induced by nuclear gene mutations include the maize iojap and nonchromosomal stripe ( NCS ) mutants (e.g., Walbot and Coe, 1979; Roussell et al., 1991; Han et al., 1992; Gu et al., 1993) and the barley albostrians mutant (e.g., Hess et al., 1994). Both iojap and albostrians are recessive and give rise to defective, maternally inherited plastids that undergo sorting out to form clonal sectors of affected (white) cells. In both mutants, the plastid defect is traceable to a loss of chloroplast ribosomes and an inability to synthesize chloroplast DNA-encod...

SPPA1 is a protease in the plastids of plants, located in non-appressed thylakoid regions. In this study, T-DNA insertion mutants of the single-copy SPPA1 gene in Arabidopsis thaliana (At1g73990) were examined. Mutation of SPPA1 had no effect on the growth and development of plants under moderate, non-stressful conditions. It also did not affect the quantum efficiency of photosynthesis as measured by dark-adapted Fv/Fm and light-adapted ΦPSII. Chloroplasts from sppA mutants were indistinguishable from the wild type. Loss of SPPA appears to affect photoprotective mechanisms during high light acclimation: mutant plants maintained a higher level of non-photochemical quenching of Photosystem II chlorophyll (NPQ) than the wild type, while wild-type plants accumulated more anthocyanin than the mutants. The quantum efficiency of Photosystem II was the same in all genotypes grown under low light, but was higher in wild type than mutants during high light acclimation. Further, the mutants retained the stress-related Early Light Inducible Protein (ELIP) longer than wild-type leaves during the early recovery period after acute high light plus cold treatment. These results suggest that SPPA1 may function during high light acclimation in the plastid, but is non-essential for growth and development under non-stress conditions.

Synthesis of carotenoids in higher plants occurs in the plastids, but all of the required enzymes are coded for in the nuclear genome and are post-transcriptionally imported into the plastid compartment. Regulation of the synthesis of the enzymes is poorly understood. The two-step desaturation of phytoene to zeta-carotene, carried out by the enzyme phytoene desaturase (PDS), is one of the earliest steps in the pathway and has been studied in several systems. Previous analyses of phytoene-accumulating tissue suggested that there may be feedback regulation of PDS gene transcription, with higher expression in white tissue. To investigate this regulation further, we examined phytoene-accumulating tissue in Arabidopsis thaliana (L.) Heynh. Two types of phytoene-accumulating tissue were studied: Norflurazon-bleached plants and white sectors from the immutans variegation mutant. Based on competitive RT-PCR measurements of PDS mRNA and immunochemical detection of PDS protein, we determined that there is no significant induction of PDS gene expression specific to white tissue, indicating that PDS expression is independent of the pigment status of the cells. Reasons why our results differ from those in other systems are discussed.